Characterization of transthyretin variants in familial transthyretin amyloidosis by mass spectrometric peptide mapping and DNA sequence analysis

Transthyretin (TTR) is a 127-amino acid residue transport protein. In plasma, TTR exists as a tetramer and binds the hormone thyroxine and the retinol-binding protein-vitamin A complex. Amino acid substitutions in TTR are hypothesized to destabilize the tetramer and cause the protein to form intermediates that self-associate into amyloid fibrils. Familial transthyretin amyloidosis (ATTR) is associated with extracellular deposition of wild-type TTR, its variants or fragments as amyloid fibrils in various tissues and organs. A definitive diagnosis of ATTR depends on the detection and identification of TTR variants. Electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS), in combination with trypsin digestion, have been shown to be powerful tools in characterizing TTR variants. Typically, TTR or its tryptic digest is analyzed by MALDI-TOF MS, liquid chromatography ESI MS, or both. Analysis of tryptic digests by MALDI-TOF MS does not provide enough sequence coverage in TTR to identify all possible modifications. To improve sequence coverage, aliquots of immunoprecipitated TTR samples were digested with trypsin, lysyl endopeptidase Lys-C, or endoproteinase Asp-N. Identification of the peptides from each digest by MALDI-TOF MS provided preliminary information about the sites and mass shifts due to amino acid substitutions from genetic mutations and to posttranslational modifications. The location and identity of the modifications in the variant proteins were then confirmed by tandem mass spectrometry, accurate mass measurements, and direct DNA sequence analysis. Using these methodologies, we achieved 100% sequence coverage. The detection of two nonpathologic variants (Thr119Met and Gly6Ser) and four pathologic variants (Phe64Leu, Asp38Ala, Phe44Ser, and previously unreported Trp41Leu) are described as illustrations of this approach.     

Fluorescence diagnostics of oil pollution in coastal marine waters by use of artificial neural networks

We discuss the problems with and the real possibilities of determining oil pollution in situ in coastal marine waters with fluorescence spectroscopy and of using artificial neural networks for data interpretation. In general, the fluorescence bands of oil and aquatic humic substance overlap. At oil concentrations in water from a few to tens of micrograms per liter, the intensity of oil fluorescence is considerably lower than that of humic substances at concentrations that typically are present in coastal waters. Therefore it is necessary to solve the problem of separating the small amount of oil fluorescence from the humic substance background in the spectrum. The problem is complicated because of possible interactions between the components and variations in the parameters of the fluorescence bands of humic substances and oil in water. Fluorescence spectra of seawater samples taken from coastal areas of the Black Sea, samples prepared in the laboratory, and numerically simulated spectra were processed with an artificial neural network. The results demonstrate the possibility of estimating oil concentrations with an accuracy of a few micrograms per liter in coastal waters also in cases in which the contribution from other organic compounds, primarily humic substances, to the fluorescence spectrum exceeds that of oil by 2 orders of magnitude and more.     

Internal hydrogen bonding,rotational isomerism and differentiation of 1,2- and 1,3-diglycerides by near infrared spectroscopy

The OH stretching modes of 1,2- and 1,3-diglycerides in dilute carbon tetrachloride solution have been investigated in the fundamental and first overtone region with a spectral resolution of about 1 cm⁻¹. The spectra of both position isomers can be interpreted in terms of three predominant configurations: one internally H-bonded and two involving no H-bonds. The fraction of H-bonded molecules appears to be relatively low. The data suggest that the H-bonded form of 1,3-diglycerides has atrans, gauche configuration around the glyceride CC bonds, while the non-H-bonded forms probably have atrans, trans configuration around the glyceride backbone, OH being eithertrans orgauche with respect to the nearest C-H group. In 1,2-diglycerides H-bonds seem to be predominantly formed between groups attached to the α, γ carbons of the glyceride residue, the configuration around the backbone beingtrans, trans; two stretching band of non-H-bonded OH groups are associated with rotational isomerism around the alcoholic CO bond. The usefulness of OH stretching modes for analyzing mixtures of 1,2- and 1,3-diglycerides is discussed. Presented in part at the AOCS meeting in Toronto, Canada, 1962. E. Utiliz. Res. & Dev. Div., ARS, U.S.D.A.     

Elevated levels of arsenic in the sediments of an urban pond: sources, distribution and water quality impacts

The discovery of significantly elevated levels of arsenic (As) in the sediments of Spy Pond motivated us to identify the principal As source and to investigate the extent of contamination within the pond. Spy Pond is located in Arlington (MA, USA), a residential and commercial town with no known history of As use by industry or agriculture. Spy Pond is a kettle-hole pond composed of two basins (north and south) separated by a shallow sill. Sediment cores from the two basins were analyzed for As content by instrumental neutron activation analysis and dated by measuring 210Pb activity. The As concentration profiles for the north and south basins had maxima that dated to approximately 1962 and approximately 1956, respectively. These dates are consistent with records of arsenical herbicide use, which indicate that between 1960-1968 sodium arsenite (NaAsO2) and arsenic oxide (As2O3) were applied to the pond to control aquatic macrophytes. Estimates of As loadings to the two basins-- approximately 410 kg ( approximately 32 kg ha(-1)) to the north and approximately 5800 kg ( approximately 580 kg ha(-1)) to the south--are consistent with the range of application rates reported for other lakes treated with arsenical herbicides. To determine the extent of As contamination in the pond, 68 surface sediment samples were analyzed by energy dispersive X-ray fluorescence. Arsenic levels ranged from 1 to 2600 ppm in the north basin (n = 49) and from 120 to 1100 ppm in the south basin (n = 19). Background sediment-As levels for the area are 10-40 ppm. The highest concentrations of As in the sediments of Spy Pond are comparable to levels measured in lakes contaminated with chemical manufacturing and mining wastes. These results are discussed with respect to As remobilization and sediment treatment measures planned for the pond.     

Electron beam irradiation for structuring of molecular assemblies

Nontraditional applications of electron beam irradiation for patterning of molecular assemblies are considered. The electron beam can have the following effects on molecular layers: destruction of molecular structure under e-beam irradiation with a successive formation of new molecular system when the irradiation is stopped; variation of the properties of the layer after e-beam irradiation; crosslinking of molecules in the layer under irradiation; modification of the templates for the successive film growth, providing different growing conditions in irradiated and nonirradiated areas; and activation of the solid support surface and molecular systems in the film resulting in the increased adhesion of the layer to the substrate in irradiated areas. All these effects were used for patterning of thin layers of different compounds. Five classes of molecular systems were considered, namely, films of simple surfactant molecules, layers of charge-transfer complexes, films of conducting polymers, aggregated nanoparticulate layers and films of nanoengineered polymeric capsules. Characteristic features of patterning processes in each particular case are discussed.     

Better use of European (regional) resources

In case of an accidental release of radioactivity and subsequent contamination of the food chain, many samples need to be collected and analysed, and this is far from being a simple issue. The determination of contamination levels requires accredited laboratories, approved and certified procedures and methods, transparency and above all prompt results, as stakeholders in general cannot afford waiting. Adequate decisions require fixed norms, stable in time, and accepted internationally. Moreover, an effective policy relies on traceability of products as well. There are huge requirements of harmonisation of procedures, traceability of data, database management, priority settings etc. Accredited laboratories tend to make use of reliable techniques but these have been optimised for low radiation levels and high accuracy for routine analyses, often in the framework of radiological surveillance of the territory, drinking waters or the food chain. It is obvious that such procedures, although very accurate and sensitive, are not suited for urgent decisions in crisis situations. Similarly, accredited analysis methodologies may start from large quantities of product in order to decrease limits of detection; however, this involves sometimes long times for drying or chemical treatment, introducing important delays. Furthermore, large quantities of samples would simply result in the saturation of the analytical capabilities of one country. Adequate actions and informed decisions during a nuclear accident will require an analytical infrastructure that individual countries do not have; hence there is a clear need to establish regional collaboration and co-operation. This paper includes an example of such collaborative work and mutual assistance, and also touches on how sharing tools for decision making, analytical resources, sample collection procedures and analysis would promote trust, reliablity in the results, a common approach toward minimizing the effects of a radiological disaster and above all unity. Last but not least, this paper also poses a challenge: Nuclear accident management implies that all responsible parties have to guarantee that decision support systems have access to data and information in the best available and consistent manner. This will not be achieved in an independent and isolated manner.     

Characterization of endothelin-1 and nitric oxide generating systems in corpus luteum-derived endothelial cells

Endothelium-derived endothelin-1 (ET-1) and nitric oxide (NO) are pivotal regulators of corpus luteum (CL) function. To have a better insight into their synthesis and action, members of the ET system (ET-1, ET converting enzyme (ECE-1) isoforms a-d, ETA and ETB receptors) along with NO synthase (NOS) isoforms--endothelial (e)NOS and inducible (i)NOS--were quantified in CL-derived endothelial cells (CLEC). The expression of these genes in microvascular CLEC, obtained by lectin-coated magnetic beads, was compared with cells removed from the luteal microenvironment and maintained in culture for different durations, and with endothelial cells (EC) derived from a large blood vessel (i.e. bovine aortic endothelial cells, BAEC). The profile of gene expression in the different EC types was determined by quantitative real-time PCR. Freshly isolated EC from mid-cycle CL exhibited high ET-1 receptor expression (both ETA and ETB), low ET-1 synthesizing ability (both prepro (pp) ET-1 and ECE-1), but elevated iNOS - the high throughput NOS isoform. The distinct phenotype of CLEC was lost soon after an overnight culture. ETA and ETB receptor levels declined, ppET-1 levels increased while iNOS was reduced. These changes were extenuated during long-term culture of CLEC. The general pattern of gene expression in BAEC and long-term cultured CLEC was similar yet some differences, reminiscent of freshly isolated CLEC, remained: ECE-1c, ETB receptor and NOS isoforms were expressed differently in BAEC as compared with lines of CLEC. This study suggests that the luteal microenvironment is necessary to sustain the selective phenotype of its resident endothelial cells. The inverse relationship between ppET-1 and iNOS observed in freshly isolated CLEC and in cultured cells is physiologically significant and suggests that ET-1 and NO may modulate the production of each other.     

Instructions for Authors

Instructions for Authors

Instructions for Authors     

Importance of the phase and amplitude in the fractional Fourier domain

The importance of the amplitude and phase in the fractional Fourier transform (FT) domain is analyzed on the basis of the rectangular signal and the real-world image. The quality of signal restoration from only the amplitude or from only the phase of its fractional FT by applying the inverse fractional FT is considered. It is shown that the signal reconstructed from the amplitude of the fractional FT usually reveals the main features of the original signal only for relatively low fractional orders. On the basis of phase information in the fractional FT domains, significant details of the signal can be obtained for nearly all fractional orders.     

Intramolecular effects on the fundamental hydroxyl stretching vibration in derivatives of fats and related compounds

The intramolecular environment of the hydroxyl group in several compounds related to fats has been investigated by infrared measurements of the fundamental stretching vibration, using a lithium fluoride double monochromator and sufficiently low concentrations (.001–.05 M) in carbon tetrachloride to avoid intermolecular effects. In saturated alcohols and saturated hydroxyl derivatives of esters, the OH stretching frequency is found to depend on the number of hydrogen and carbon atoms which form the immediate environment of the OH group. Since this varies with rotational isomerism about the C-O single bond, the infrared hydroxyl band is composite, made up of components whose strengths give information about the relative amount of each rotational isomer present. In unsaturated alcohols and unsaturated hydroxy-esters, rotational isomers permitting hydrogen bonding to π-electrons are stabilized according to the accessibility of the unsaturated link to the hydroxyl group, thus altering the proportions of the rotational isomers. Presented at the AOCS meeting in Toronto, Canada, 1962. Eastern Utiliz. Res. & Dev. Div., ARS, U.S.D.A.